<Abstract Centered> an ABSTRACT of the THESIS OF

AN ABSTRACT OF THE THESIS OF

Julie L. Colvin for the degree of Master of Science in Food Science and Technology presented on June 8, 2018.

Title: Differences in Taste Responses between Tongue Regions

Abstract approved:

______Juyun Lim

Due to a combination of misinterpretation and misleading illustration, the premise of a

“tongue map”, which indicated that sweetness could only be detected at the front of the tongue and bitterness could only be detected on the back, became wide spread. In fact, all taste qualities can be detected on the front, back and sides of the tongue. Studies on regional differences within the oral cavity have typically reported differences in taste response depended on taste quality. Most of these studies have had subjects keep their tongue still or mouth open while evaluating samples to prevent the spread of stimuli to other regions of the oral cavity. However, tongue and mouth movements are naturally paired with taste perception in normal eating situations. This intraoral movement can cause stimuli to spread throughout the mouth and may have additional effects on taste perception. Therefore, studying the effects of intraoral tongue and mouth movements may be important to understanding taste perception mechanisms. Notably, some reports have not specified the tasting mode used in their study. In addition, it has been suggested that maltooligosaccharides (MOS) may be detected independently from the classic sweetness receptor, but it is unknown whether regional responsiveness differs between sucrose and MOS. The current study was designed to investigate 1) the effects of taste quality on regional differences in responsiveness between the front and back of the tongue, and 2) the effects of “passive” and “active” tasting modes on relative regional differences in taste responsiveness. Along with the two carbohydrates tested (i.e. sucrose and MOS), quinine and MPG were also included to represent bitter and umami taste qualities, respectively. In the passive tasting condition, the front of the tongue was found to be more responsive to both carbohydrates, but no regional differences were seen for quinine or MPG. In the active tasting condition, the back of the tongue was more responsive to quinine and MPG, but no differences were found for either carbohydrate.

These findings indicate that there are regional differences in taste responsiveness between the front and back of the tongue, and that they are dependent on taste quality and modulated by tasting mode. Further, the effects of tasting mode were taste quality dependent only on the back of the tongue. This indicates that interactions between taste and intraoral movements may be different between tongue regions.

Differences in Taste Responses between Tongue Regions

by Julie L. Colvin

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented June 8, 2018 Commencement June 2018

Master of Science thesis of Julie L. Colvin presented on June 8, 2018.

APPROVED:

______Major Professor, representing Food Science and Technology

______Head of the Department of Food Science and Technology

______Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

______Julie L. Colvin, Author

ACKNOWLEDGEMENTS

First, I would like to express my sincere appreciation to my major advisor, Dr.

Juyun Lim, for allowing me the opportunity to join this lab. Her work ethic and passion for science inspired me to pursue research, and her expertise and mentorship have helped me grow as a researcher, writer, and scientist. I would also like to thank my committee members, Dr. Michael Penner, Dr. Elizabeth Tomasino and Dr. James Hermes, for their feedback on the present work and commitment of time as part of my defense committee.

Further, I would like to thank my current and former lab mates, Alex Pullicin,

Tyler Flaherty, Trina Lapis, Tyler Linscott, Amy Balto, Rachel Silver, and Erin Schenk, for helping me grow as a researcher and for making my experience in graduate school more fun. To the students, staff, and faculty of the Food Science and Technology

Department, thank you for your help, guidance and support throughout my time here at

OSU. I would also like to acknowledge Sue Queisser, Aimee Hasenbeck, and the others who I worked with in the consumer lab, thank you for your support and insights into food science and other topics. I would also like to thank my friends, those at OSU and not, who have been there for me when it was time to step out of the lab, library, and classroom for a while. Finally, I would like to dedicate this work to my family. Thank you for your unwavering support during my time in this program.

CONTRIBUTION OF AUTHORS

Julie Colvin wrote Chapters 1 and 3. Julie Colvin performed the data collection for

Chapter 2. Julie Colvin, Juyun Lim, and Alexa Pullicin were involved with the research design and data analysis of Chapter 2. Chapter 2 was written by Julie Colvin and Juyun

Lim with inputs by Alexa Pullicin.

TABLE OF CONTENTS

Page

1. General Introduction ...... 1

1.1 Functions of the Gustatory System in Humans ...... 1

1.2 Peripheral Anatomy of the Human Gustatory System ...... 2

1.2.1 Papillae ...... 3

1.2.2 Taste Buds ...... 4

1.2.3 Taste Receptors ...... 5

1.2.4 Nerves ...... 6

1.2.5 Dysfunction of the Peripheral Gustatory System ...... 6

1.3 Previous Studies on Regional Differences in Taste Response ...... 9

1.3.1 Tongue Maps: Misinterpretations and Clarifications ...... 9

1.3.2 Regional Differences in Taste Sensitivity ...... 10

1.3.3 Regional Differences in Taste Responsiveness ...... 11

1.3.4 Stimuli Concentration ...... 12

1.3.5 Umami Stimuli ...... 13

1.4 Regional Differences for Alternative Tastes ...... 15

1.4.1 Taste of Fat ...... 15

1.4.2 Taste of Complex Carbohydrates ...... 16

1.5 The Effects of Intraoral Movement on Taste Perception ...... 18

1.5.1 Intraoral Processing ...... 19

1.5.2 Stimuli Spread ...... 20

1.5.3 Other Effects of Intraoral Movements ...... 21

1.6 Study Objectives ...... 22

TABLE OF CONTENTS (CONTINUTED)

Page

2. Regional Differences in Taste Responsiveness: Effects of Taste Quality and Tasting Mode ...... 24

2.1 Abstract ...... 26

2.2 Introduction ...... 27

2.3 Materials and Methods ...... 30

2.3.1 Subjects ...... 30

2.3.2 Stimuli ...... 31

2.3.3 Experimental procedure ...... 33

2.3.4 Data analysis ...... 35

2.4 Results ...... 36

2.4.1 Experiment 1: passive tasting ...... 36

2.4.2 Experiment 2: active tasting ...... 38

2.4.3 Impact of tasting mode on regional differences in taste responsiveness across stimuli ...... 38

2.5 Discussion ...... 39

2.5.1 Regional Differences in Taste Responsiveness: Taste Quality ...... 40

2.5.2 Regional Differences in Taste Responsiveness: Tasting Mode ...... 43

2.5.3 Regional Taste Responsiveness to Carbohydrate Stimuli ...... 45

2.6 Summary ...... 46

2.7 References ...... 47

3. General Conclusions ...... 50

Bibliography ...... 53

LIST OF FIGURES

Figure Page

1. Diagram of taste papillae and cranial nerves ...... 3

2. Taste Bud Diagram ...... 4

3. Chemotopic tongue maps from the classic study of Hänig...... 9

4 Psychophysical functions on the front and back of the tongue ...... 12

5. Diagram of the four target locations on a tongue ...... 32

6. Picture of the front and back of the tongue being swabbed ...... 34

7. The mean log perceived intensities of stimuli sampled on the front and back using a passive tasting mode ...... 37

8. The mean log perceived intensities of stimuli sampled on the front and back using an active tasting mode ...... 39

9. The mean differences in log perceived intensities between passive and active tasting modes ...... 44

CHAPTER 1

GENERAL INTRODUCTION

1.1 FUNCTIONS OF THE GUSTATORY SYSTEM IN HUMANS

The gustatory system is composed of taste receptors which detect chemical compounds in the oral cavity, afferent cranial nerves which relay information to the brain, and the gustatory cortex, part of the central nervous system which processes and integrates sensory information (see reviews de Araujo and Simon, 2009; Niki et al.,

2010). Both of the former are parts of the peripheral nervous system. The major output of this system is salient taste sensation, which, in addition to making food consumption enjoyable (Scott, 2005), is believed to be indicative of the potential nutritive value and the relative safety of food (Lindemann, 2001; Reed and Knaapila, 2010; Breslin, 2013); both of these are factors in ingestive behavior. Taste also triggers the preparative release of digestive enzymes and hormones prior to ingestion (Teff et al., 1991; Abdallah et al.,

1997; Just et al., 2008).

Taste sensations are often classified as sweet, umami, salty, bitter, and sour. They are generally elicited by metabolically relevant classes of chemicals including sugars, amino acids, salty, alkaloids, and acids, respectively (Frank and Hettinger, 2005). Each of these taste qualities is believed to be associated either with beneficial food components

(i.e. sugar, protein, and electrolytes) or potentially toxic compounds (i.e. poisons or spoilage products), which are palatable or aversive, respectively (Breslin and Spector,

2008). Although the five prototypical taste qualities (i.e., sweet, umami, salty, bitter, and sour) have been widely accepted, other unique taste qualities have been proposed to be 2 elicited by other chemicals including: free fatty acids (Laugerette 2005; Mattes 2009b,

2009a; Haryono et al. 2014; Running et al. 2015), glucose oligomers (Lapis et al., 2016;

Pullicin et al., 2017), water (Gilbertson et al., 2006; Zocchi et al., 2017), and calcium

(Maruyama et al., 2012).

In summary, the functions of the gustatory system are 1) sensory discrimination between stimuli on the basis of taste quality or intensity, 2) quality assessment or hedonic judgement of food, and 3) physiological responses occurring prior to consumption which aid in digestion. These functions are critical for health and survival, which may be why it is relatively rare to experience complete taste loss; it has been proposed that redundancies in the gustatory system may function to protect against complete taste loss (Tomita et al.,

1986). However, taste loss for specific taste qualities (specific aguesia), taste insensitivity

(hypoaguesia), phantom taste (phantaguesia) and taste abnormality (dysgeusia), are seen as a result of surgery, head trauma, ear infections, genetics, aging, medications and other underlying medical conditions (Gent et al., 1987; Lugaz et al., 2002; Goins and Pitovski,

2004; Boyce and Shone, 2006; Douglass and Heckman, 2010; Windfuhr et al., 2010;

Bartoshuk et al., 2012; Pelletier et al., 2013; Doty et al., 2016; Snyder and Bartoshuk,

2016).

1.2 PERIPHERAL ANATOMY OF THE HUMAN GUSTATORY SYSTEM

Taste sensations occur when chemical compounds, typically those with some kind of metabolic or health consequences, come into contact with microvilli at an apical opening of taste buds, which contain a variety of taste receptors (Hoon et al., 1999;

Chaudhari and Roper, 2010). Cranial nerves innervate taste receptors and relay gustatory

3 information to the central nervous system (i.e. gustatory cortex; de Araujo and Simon,

2009; Purves et al. 2001). Taste receptors are innervated (i.e., supplied with nerves) by branches of one of three cranial nerves: facial (VII), glossopharyngeal (IX), or vagus (X).

1.2.1 Papillae

There are three major types of papillae in the mouth which are involved in the detection of taste: fungiform, foliate, and circumvallate papillae (see Fig. 1) (Purves et al., 2001; Chandrashekar et al., 2006; Chaudhari and Roper, 2010). Other papillae on the tongue include the filiform papillae, which are located over the entire dorsal surface of the tongue, respond to tactile stimulation (Kawasaki et al., 2012) and are innervated by the trigeminal nerve (Snyder and Bartoshuk 2016). Fungiform papillae are mushroom- shaped epithelial structures located mainly on the front of the tongue. Papillae on the palate are structurally similar to the fungiform papillae (Nilsson, 1979b; Imfeld and

Figure 1 Diagram of taste papillae and cranial nerves which innervate the tongue. Mouth and tongue image by Alexa Pullicin. Taste buds adapted from Chandrashekar et al. 2006.

Schroeder, 1992; Ikeda et al., 2002). Foliate papillae are located in folds on the edges of the back of the tongue. And circumvallate papillae are raised round structures surrounded by grooves and moat-like fissures (Purves et al. 2001 Chandrashekar et al., 2006;

Chaudhari and Roper, 2010). Humans typically have 9 circumvallate papillae arranged in a V or Y shaped pattern, but some have reported the number ranging from 3-14 (Spuhler,

1950).

1.2.2 Taste Buds

Taste buds are embedded within papillae in the oral cavity. Taste buds contain between 50-100 taste receptor cells bundled in an onion shape with an opening at the apex (i.e., a taste pore) which allows for interaction between taste receptors microvilli and chemical stimuli (i.e. taste stimuli) present in the oral cavity (see Fig.

2) (Hoon et al., 1999). Fungiform papillae contain approximately 1-3 taste buds on average; however, individuals can have between Figure 2 Taste bud diagram. Adapted from Chandrashekar et al. 0-9 taste buds per fungiform papillae (Purves et 2006. al. 2001; Arvidson, 2007). Of taste buds within fungiform papillae, 87% of are located on the anterior 2 cm of the tongue (Cheng and Robinson, 1991). Foliate and circumvallate papillae each contain hundreds of taste buds (Purves et al. 2001).

1.2.3 Taste Receptors

Taste receptor cells (see Fig. 2) are responsible for the detection of certain chemicals within the oral cavity. G protein-coupled receptors (GPCR) mediate the transduction of sweet, bitter, and umami tasting substances; in contrast, ion channels are responsible for the detection of sour and salty taste stimuli. The T1R2/T1R3 is considered the major sweetness receptor (Chaudhari and Roper, 2010). There are approximately 30

T2R bitter receptors, some of which detect multiple bitter compounds, and others which are stimuli specific (Chandrashkar et al. 2006). And there are at least 3 proposed receptors for umami taste: T1R1/T1R3, mGluR1, and mGluR4 (Chaudhari and Roper,

2010; Roper, 2013).

There is evidence that receptor cells are differentially distributed across tongue regions (Mattes, 2009b; Bachmanov et al., 2014). In other words, while taste buds contain a variety of taste receptor cells, not all taste buds necessarily contain the same receptor cells. This may contribute to differences in regional taste responses. In mouse and rat tongues, T2R receptors, associated with bitter taste perception, were expressed more frequently in the circumvallate than the fungiform papillae; the opposite was found for T1R1 receptors, which are associated with sweet and umami taste perception (Hoon et al. 1999). Further, the location of taste receptors within the taste bud may also modulate sensitivity; the closer to the taste pore the more active a taste receptor may be (Hoon et al.

1999).

1.2.4 Nerves

Taste receptors on the anterior (front) portion of the tongue are innervated by the chorda tympani (a branch of cranial nerve VII), while the trigeminal nerve (cranial nerve

V) transmits touch from this region; the posterior (back) third of the tongue is innervated by the glossopharyngeal nerve, which relays both taste and tactile information (see Fig.

1) (Purves et al., 2001; de Araujo and Simon, 2009; Niki et al., 2010). Taste information from the soft palate (the posterior 1/3rd of the roof of the mouth), is primarily transduced by the greater superficial petrosal branch of the facial nerve (VII). The hard palate (the anterior 2/3rds of the roof of the mouth) is innervated by a branch of the vagus nerve

(cranial nerve X; Ikeda et al., 2002); however, hard palate is largely insensitive to taste

(Green and Nachtigal, 2012), except at relatively high concentrations of stimulus

(Nilsson, 1979a).

It has been hypothesized that cranial nerves which innervate the tongue have distinct functions (Breslin, 2001; Frank and Hettinger, 2005). Specifically, the chorda tympani nerve may relay quality-specific responses and preference, while the glossopharyngeal nerve may be more associated with reflexive actions, such as aversion or gagging in response to bitterness. The strongest evidence for this hypothesis is in nerve transection studies in animals (Spector 2003). Variations in taste response may also arise from taste- or stimuli- specific nerve fiber sensitivity (Frank et al., 2008).

1.2.5 Dysfunction of the Peripheral Gustatory System

Few differences are seen physiologically or perceptually between the left and right sides of the tongue in healthy subjects (Kroeze, 1979; McMahon et al., 2001). But if

7 either one or both cranial nerves that innervate the tongue are damaged, it can result in regional taste loss. This can affect taste perception and health (Goins and Pitovski, 2004;

Bartoshuk et al., 2012). Surprisingly, nerve damage does not always result in noticeable changes in taste sensation in normal eating situations (Snyder and Bartoshuk, 2016). This is often attributed to a combination of 1) central inhibition between the glossopharyngeal nerve and chorda tympani nerve, and 2) the referral of taste sensation to locations being stimulated by touch. Due to mutual inhibition between gustatory nerves in the central nervous system, when one nerve (e.g. right chorda tympani) is damaged or anesthetized it results in disinhibition of the contralateral nerve on the opposite region of the tongue (e.g. left chorda tympani) and a subsequent increase in taste sensitivity on this region

(Catalanotto et al., 1993; Bartoshuk et al., 2005). Regional losses of taste sensation are masked by “tactile capture,” which occurs when taste is referred to a region of the tongue when it is touched (Todrank and Bartoshuk, 1991). To demonstrate this, a swab was swept across the anterior portion of a healthy, unanesthetized tongue. Taste sensation increased in intensity from the first edge to the tongue tip, as expected. However, the increased taste sensation experienced on to the tip of the tongue was carried over, or

“captured”, by the tactile stimulation of the swab. Therefore, the perceived taste sensation remained elevated when the swab contacted the opposite edge of the tongue. Other experiments have shown similar referral of taste sensation to the location of touch stimulation (Green, 2003; Lim and Green, 2008).

Overall, mild damage can increase regional and whole mouth taste sensation, but if both nerves are damaged or an individual was insensitive to taste prior to injury,

8 decreases in overall taste sensitivity may be observed (Bartoshuk et al., 2012).

Interestingly, those who are most sensitive to taste (i.e. supertasters) are more likely to notice the effects of cranial nerve damage and have more severe symptoms, such as

Burning Mouth Syndrome (Grushka, 1987). The compensatory tactile mechanism discussed above would be particularly effective at masking taste loss during normal oral processing (e.g. mastication or bolus formation) due to the repetitive tactile stimulation that may result from contact with the food object and/or tongue and mouth movements.

The complexity and variability of the consequences of cranial nerve damage led

Bartohsuk (1989) to the development of the “Spatial Taste Test” for the diagnosis of taste damage; this test compares the perceived regional intensities of stimuli to those of a

“normal” group. Large discrepancies, or discrepancies on specific regions or for specific taste qualities, are considered indicative of abnormal taste perception. In this method, one rating is taken from each region (i.e., fungiform, foliate, and circumvallate, see below); the right and left side of the tongue are swabbed sequentially and one intensity rating is made on a 9-point hedonic scale. Whole mouth ratings (with swallowing) are then taken to compare against regional taste responsiveness. Because of the variability in the outcomes of cranial nerve damage and the compensatory mechanisms which mask it, regional testing is needed to fully understand the extent and consequences of cranial nerve damage. It follows that understanding how different oral regions contribute to and affect taste perception may also, in part, require testing on discrete tongue regions.

1.3 PREVIOUS STUDIES ON REGIONAL DIFFERENCES IN TASTE RESPONSE

1.3.1 Tongue Maps: Misinterpretations and Clarifications

Early studies on regional differences were trying to prove the existence of independent physiological processes for the detection of “basic” or “primary” taste qualities (Boring, 1942; Erickson,

2008). Chemotopic renderings of the surface of the tongue drawn by Hänig

(1901) (see Fig. 3) are the first known Figure 3 Chemotopic tongue maps from the classic study of Hänig (1901). The density examples of tongue maps being used to of dots is representative of the sensitivity (inverse of threshold detection) of the display regional differences in taste tongue region to a taste quality. Translated by Lindemann (1999). responses (Lindemann, 2001). While regional differences in taste responses were explored prior to the development of tongue maps (Boring, 1942), the tongue map is the most well-known example of regional differences in taste responses. Sensitivity of tongue regions to each taste are illustrated by the density of dots on each region; higher density correlates with higher sensitivity. These maps indicate that the tongue is most sensitive to sweetness on the tip, bitterness on the back, and sourness and saltiness on the sides.

It is important to note that tongue maps have often been incorrectly interpreted to illustrated tongue regions which are independently responsible for the perception of distinct taste qualities (Breslin, 2001; Lindemann, 2001). For example, Hänig (1901)

10 found the back of the tongue was most sensitive to bitter taste, however this does not mean that bitterness was undetectable on other areas of the tongue. The misinterpretation of the original “tongue map” has been attributed, in part, to a mistranslation from the original German manuscript (Bartoshuk and Beauchamp, 1994). Closer inspection of the works of Boring and Hänig revealed that neither used numeric or otherwise quantifiable scales to present sensitivity data, which may have further contributed to confusion

(Breslin, 2001). Despite the controversy surrounding the misinterpretation of the tongue map, studies have continued to find differences in taste responses across the tongue and throughout the oral cavity (Collings, 1974; Nilsson, 1979a; Matsuda and Doty, 1995;

Yamaguchi and Ninomiya, 2000; Sato et al., 2002; Green and Schullery, 2003; Green and

George, 2004; Warnock and Delwiche, 2006; Grover and Frank, 2008; Green and

Nachtigal, 2012; Feeney and Hayes, 2014; Calviño, 2016; Doty et al., 2016)

1.3.2 Regional Differences in Taste Sensitivity

A later study by Collings (1974) reaffirmed that sweet, bitter, salty, and sour taste qualities could be detected on multiple gustatory regions. Collings also reported patterns of regional taste sensitivities, in terms of quality recognition threshold (i.e. concentration at which a subject can correctly identifying sucrose as “sweet”), which were similar to those reported by Hänig (1901). Dissimilarly, the tip of the tongue was found to be more sensitive to quinine hydrochloride and urea (i.e. bitter tasting stimuli) than the back; the soft palate and was found to be more sensitive to bitter taste than any tongue region.

Other studies have also found the soft palate to be relatively sensitive to sweet (Ikeda et al., 2002), umami (Satoh-Kuriwada et al 2012) and bitter (Nilsson, 1979a) tastes.

However, these findings at threshold levels of stimuli are not necessarily applicable to investigations of regional differences in responsiveness to higher concentrations (i.e. suprathreshold levels) of stimuli (Keast and Roper, 2007; Webb et al., 2015).

1.3.3 Regional Differences in Taste Responsiveness

Regional differences in taste responsiveness (i.e. responses to suprathreshold taste solutions) have generally been found to be dependent on taste quality; one exception reported the posterior of the tongue was more responsive to all stimuli tested: sucrose, quinine hydrochloride, NaCl and citric acid (Grover and Frank, 2008). The authors hypothesized that this may have been due to stimulus application method (i.e. cotton tipped swabs) which may have stimulated the taste buds in the grooves of the circumvallate papillae more, resulting in higher taste intensities. While this seems unlikely due to the fact that most of the other regional studies in responsiveness have also used cotton tipped swabs, the potential for discrepancies in findings of regional differences due to the method of stimulation has been noted (Nilsson, 1979a).

In other studies using sucrose, sodium chloride, and citric acid to elicit sweet, salty and sour tastes respectively, no significant regional differences in responsiveness have been reported (Green and Schullery, 2003; Green and George, 2004; Green and

Nachtigal, 2012; Feeney and Hayes, 2014) with one exception (i.e. salty was more intense on the front of the tongue; Green and Nachtigal, 2012). Bitter taste elicited by quinine has generally been reported as more intense on the back of the tongue, although the degree of difference has varied (Green and Schullery, 2003; Green and George, 2004;

Feeney and Hayes, 2014); this may suggest that the degree of difference in

12 responsiveness between regions is concentration dependent within suprathreshold level stimuli (see Stimuli Concentration below). The back of the tongue has also been reported to be markedly more responsive to umami/savory taste stimuli than the front of the tongue in all studies (Yamaguchi and Ninomiya, 2000; Green and Nachtigal, 2012;

Feeney and Hayes, 2014).

1.3.4 Stimuli Concentration

Collings (1974) noted that previous experiments had not used suprathreshold level stimuli. Therefore, in addition to taste quality recognition thresholds, Collings measured the psychophysical functions (i.e., the growth of taste sensation magnitude in response to

increases in concentration) of each

taste stimulus across all oral loci. It

was found that the slopes of

psychophysical functions differed

between oral loci. For example, the

intensities of bitter stimuli (i.e. urea

and quinine) increased more

quickly on the back of the Figure 4 Psychophysical functions on the front and back of the tongue. Magnitude of taste sensation of tongue than the front (see Fig 4). quinine hydrochloride as concentration (molar) increases on the front (solid line) and back (dotted These results indicated the line) of the tongue. Modified from Collings (1979a). degree of difference in responsiveness between regions may increase with stimuli concentration. A similar trend was seen for sodium chloride. Regional differences for sucrose may also be concentration

13 dependent. Nilsson (1979) reported that regional differences in responses to sucrose between tongue regions were higher at relatively low concentrations when measured in terms of taste quality recognition threshold (i.e. concentration at which a subject can correctly identify sucrose as “sweet”). This is approximately the inverse of the relationship seen for quinine, indicating the effects of concentration are taste quality dependent. Speculatively, the concentration effects across regions may be calibrated to serve some adaptive purpose. The tip of the tongue may have relatively enhanced responsiveness to sugars, especially at somewhat weak intensities, to encourage consumption; the back of the tongue may have relatively enhanced responsiveness to bitterness at high concentrations to serve as a final warning prior to swallowing.

Taken together, these findings demonstrate that relative regional differences are concentration dependent, and that the effects of concentration are dependent on taste quality. This variation in responsiveness may serve some adaptive purpose. Therefore, intensity matching stimuli may facilitate comparisons across taste stimuli.

1.3.5 Umami Stimuli

The studies mentioned above used prototypical taste stimuli to represent the canonical taste qualities, such as sucrose and quinine hydrochloride for sweet and bitter taste, respectively. Generally, the use of standard prototypical taste stimuli allows for better comparisons across studies. However, it should be considered that some taste stimuli that fit into the basic taste categories may: elicit multiple taste qualities (e.g. MSG elicits salty and umami tastes; Chen et al. 2009; Linscott and Lim 2016; Green et al

2016), activate different taste receptors (e.g. bitter compounds can have independent or

14 multiple transduction mechanisms; Delwiche et al., 2001b), or elicit different spatial profiles (e.g. sucrose and aspartame: Breslin, 2001; Warnock and Delwiche, 2006). All of these may affect findings of regional differences and could subsequently elucidate differences in taste perception mechanisms between regions.

Notably, the studies on regional differences for umami/savory taste have used either monosodium glutamate (MSG) or a mixture of MSG and inosine monophosphate

(Feeney and Hayes 2014; Green and Schullery 2003; Yamaguchi and Ninomiya 1999).

The use of MSG as the prototypical tastant for umami taste is surprising considering the well-documented salty taste of MSG (Maryuama et al. 2006; Chen et al, 2009; Linscott and Lim 2016; Green et al. 2016). Some have reported that this can cause taste quality confusion (Green et al 2016; Mueller et al. 2011). For example, in one study up to 28% of subjects misidentified MSG as “salty” tasting, rather than umami (Mueller et al. 2011).

Taste quality confusion may also be more likely on the tip of the tongue, given that the back of the tongue has been shown to discriminate MSG and other taste qualities better than the front of the tongue in mice (Ninomiya and Funakoshi, 1989). Evidence supporting this is also found in receptor studies in mice. The T1R1/T1R3 receptors, which function primarily in behavioral discrimination, are expressed primarily on the anterior tongue, while mGluRs, which discriminate umami tastes, are located on the posterior tongue (Yasuo et al., 2008). In humans, psychophysical data demonstrated that the “certainty” of taste quality recognition for MSG is higher on the back of the tongue

(Yamaguchi and Ninomiya, 2000). This may also support this hypothesis; however, it is

15 not clear whether ratings of “degree of certainty” that a taste quality was present were representative of the perceived intensity or simply the salience of taste on each region.

It is not known whether monopotassium glutamate (MPG), an umami tasting stimuli without significant salty side taste (Linscott and Lim 2016; Green et al 2016) would demonstrate a pattern of regional differences similar to those seen using MSG.

However, it has been hypothesized that it may be detected through a different transduction or receptor binding mechanism (Green et al. 2016). Assessing the regional differences in responsiveness to MPG may contribute to the growing body of research on alternate glutamate taste receptors (Maruyama et al., 2006; Yasuo et al., 2008; Chaudhari et al., 2009; Jyotaki et al., 2009; Green et al., 2016).

1.4 REGIONAL DIFFERENCES FOR ALTERNATIVE TASTES

Investigations into regional responsiveness can reveal regional variations in taste perception mechanisms (Warnock and Delwiche, 2006; Grover and Frank, 2008). This can guide more advanced techniques which are used to understand taste transduction mechanisms, such as in situ hybridization (Hoon et al. 1999). Regional differences in taste response can also help to understand the taste perception mechanisms for non- canonical taste qualities (i.e., free fatty acid taste; Mattes 2009).

1.4.1 Taste of Fat

Evidence supporting the existence of a taste for fat or “oleogustus” (Running et al., 2015) has been accumulating for both humans and rodents over the last decade or more (Mattes, 2009a; Besnard et al., 2016). One proposed receptor is the CD36 fatty acid transporter, which has been shown to be involved in the oral detection of long-chain fatty

16 acids in rats and mice; it is expressed in the apical area (i.e., near the taste pore) of the circumvallate papillae on the posterior tongue of the rat (Laugerette, 2005). A variety of receptors, including the later, have been proposed as being responsible for the detection of specific chain lengths of fatty acids in the absence of texture cues, suggesting there may be regional differences in responsiveness between stimuli; from studies on the expression patterns and ligand specificity of these receptors, the circumvallate papillae on the back of the tongue appears to be the area which would likely be most responsive to fatty acids of most chain lengths (Mattes, 2009b). Surprisingly, the results of a region study showed that all stimuli were perceived as being most intense at the fungiform papillae, followed by the circumvallate and foliate papillae, respectively. The authors hypothesize that this discrepancy may be due to inaccuracies in the proposed receptors systems or ligand specificity models for receptors; the authors also consider non-specific detection mechanism (e.g. intracellular signaling) which may be altering results. Either way, this study demonstrates the importance, and complexity, of conducting studies on human taste perception in the search for and understanding of novel taste receptor mechanisms.

1.4.2 Taste of Complex Carbohydrates

It was previously thought that complex carbohydrates did not elicit a salient taste in humans. However, studies had found that rodents perceive sucrose and Polycose (i.e., a mix of glucose oligomers and polymers) qualitatively different based on consumption

(i.e., preference) and aversion (i.e., discrimination) tests (Feigin et al., 1987; Nissenbaum and Sclafani, 1987). Nissenbaum and Sclafani (1987) hypothesized the existence of a

17 novel taste transduction mechanism for the detection of Polycose. However, it was thought that the mechanism of detection for Polycose in rodents was not present in humans (Feigin et al., 1987). Lapis et al. (2014; 2016) determined that a similar

T1R2/T1R3 independent mechanism was likely present in humans using samples which were representative of starch hydrolysis products, such as maltooligiosaccharides (MOS).

MOS occur naturally during consumption of complex carbohydrates as a result of mechanical (i.e. chewing) and enzymatic (i.e. salivary α-amylase) digestion in the mouth.

In the 2016 study, a series of MOS samples with an average degree of polymerized (DP) of 7, 14, and 44 (Balto et al., 2016) were presented to subjects. Samples were presented by rolling a cotton tipped swab across the tip of the tongue to control for texture cues.

Further, the breakdown of glucose oligomers to sugars by salivary the α-amylase in the oral cavity was inhibited using acarbose, an α-glucosidase inhibitor (Clissold and

Edwards, 1988; Balfour and McTavish, 1993). The sweetness receptor, the T1R2/T1R3 heterodimer, was inhibited using lactisole, a T1R3 inhibitor (i.e. a sweetness inhibitor;

Jiang et al., 2005). Subjects were found to be able to detect MOS (average DP 7 and 14) even when acarbose and lactisole were used. From these findings, it was concluded that glucose oligomers may also be detectable by humans through a T1R2/T1R3 independent mechanism.

Further, Lapis et al (2016) demonstrated that MOS (average DP 14) were perceived to be qualitatively different from simple sugars; samples were identified as having a “starchy” (e.g. bread- or rice- like) taste, rather than sweet taste quality. More recent studies focusing only on low-DP MOS have shown that humans can detect DP 4-7,

18 but that DP 3 (i.e., maltose) detection is dependent on the classic sweetness receptor

(Pullicin et al., 2017). While the transduction mechanisms for glucose oligomer detection are currently unknown, one study in mice indicated the glossopharyngeal nerve transection reduces appetite for Polycose more than for sucrose (Vigorito et al., 1987).

When the chorda tympani was transected, reductions in Polycose and sucrose appetite were similarly reduced. Overall, these results suggest that the detection of Polycose appetite may be more strongly mediated by the glossopharyngeal nerve, at the back of the tongue, than sucrose appetite.

To determine whether the detection of glucose oligomers in humans may also be preferentially associated with the posterior region of the tongue, the current study compared the perceived intensity of MOS and sucrose on the front and back of the tongue

(innervated by the chorda tympani and glossopharyngeal, respectively). This preliminary investigation may contribute information about the location or function of the

T1R2/T1R3 independent mechanism or elucidate potential species differences. Based on the findings previously discussed (Vigorito et al., 1987) it was hypothesized that the patterns of regional differences for MOS would be different from those for sucrose. More specifically, it was thought that MOS would be perceived as more intense on the back of the tongue than the front of the tongue. Other findings may be indicative of species differences or the effects of oral enzymatic hydrolysis (which was not controlled for in animal studies).

1.5 THE EFFECTS OF INTRAORAL MOVEMENT ON TASTE PERCEPTION

Few studies have looked at the regional effects of intraoral tongue and mouth movement on taste perception. Some studies on regional differences have instructed subjects to refrain from touching the tongue to the mouth to prevent the spread of stimuli to non-target regions (i.e., “passive” tasting mode; Collings, 1974; Nilsson, 1979a; Sato et al., 2002; Doty et al., 2016), more recent studies have not specified whether subjects were allowed to touch their tongue to other oral regions (Green and Schullery, 2003;

Green and George, 2004; Feeney and Hayes, 2014). It is currently unclear what effects intraoral movement may have on relative regional differences or how the effects may vary between regions.

1.5.1 Intraoral Processing

During normal consumption, food is placed on the tip of the tongue and taken into the mouth. A variety of intraoral manipulations, including the compression of the tongue

(and food object) against the palate, subsequently transfer food to the posterior portion of the mouth. If the food is solid, it will be chewed and swallowed; semi-solid and liquid food are more variable, but still involve complex tongue and mouth movements (de Wijk et al., 2003, 2011). Intraoral movements are highly complex, and there is a wide range of individual variation. However, the natural tasting motion of an individual has been shown to enhance sensory attributes (e.g. flavor/taste) more than any standardized approximation of mouth movement (de Wijk et al., 2003). Prior to swallowing, the tongue and palate make periodic contact (Matsuo et al., 2005), which may spread stimuli between these regions during tasting. Further understanding of the effects of tongue and

20 mouth movements is necessary to gain a better understanding of regional contributions to taste perception (Gibson 1966; Green and Nachtigal, 2012).

1.5.2 Stimuli Spread

Stimuli spread during intraoral movement could occur as a result of salivary flow or contact between the tongue and other regions of the mouth (see Intraoral Processing above). As the tongue moves, food or stimuli may spread from the dorsal surface of the tongue to the corresponding region on the palate. This could result in spatial summation, which is a proportional increase in the intensity of stimuli in response to increased stimulation area (Smith, 1971; Delwiche et al., 2001a). In this case, the stimulated area may double. The effectiveness of spatial summation has not been evaluated on the back of the tongue per se. However, Green and Nachtigal (2012) found that spatial summation likely occurred between the tongue and palate across the midline of the tongue on both the front and back of the tongue. On the back of the tongue, taste may have been enhanced as stimuli spread to the taste sensitive soft palate (Collings 1974; Nilsson

1979a; Ikeda 2002; Satoh-Kuriwada et al. 2012). In contrast, the hard palate has been reported to be largely insensitive to even high concentrations of taste (Nilsson 1979a;

Green and Nachtigal 2012). Considering the relative taste responsiveness of the hard and soft palate, it would follow that spatial summation may be greatest on the back of the tongue. While Green and Nachtigal (2012) did not find any regional differences in the effects of tongue and mouth movements, subjects were allowed to swallow taste stimuli which may have caused stimuli to spread from the front of the tongue and hard palate to more sensitive posterior regions, including the soft palate.

1.5.3 Other Effects of Intraoral Movements

The idea that there may be regional differences in the interactions between taste and intraoral movements is especially intriguing considering taste and touch signals are transduced by separate nerves on the front of the tongue (i.e., chorda tympani and trigeminal) and one nerve on the back of the tongue (i.e., glossopharyngeal). This may lead to differences in integration of taste and repetitive touch sensation, which can be generated by intraoral movements, when relayed to the central nervous system (de Araujo and Simon, 2009). Further, these differences may serve some adaptive purpose. For example, the back of the tongue may be more responsive to mouth movement because food will rarely stimulate the back of the tongue independent of tongue mouth movements. In contrast, the front of the tongue may be more sensitive prior to tongue and mouth movements, similar to how food may be sampled prior to being placed in the mouth. Green and Nachtigal (2012) found that the perceived intensity of umami taste was enhanced by tongue and mouth movement on the front and back of the tongue, while no such patterns were seen for other taste stimuli. The authors hypothesized that this may be indicative of a specific, perceptual enhancement of umami taste by tongue and mouth movements. From a functional stand point, tongue and mouth movements may be more important for umami taste due to the fact that savory foods may typically require more intraoral manipulation, as many are solids (e.g., cooked meat; Breslin 2013). It is unclear whether similar interactions may occur for other umami/savory stimuli (e.g., MPG). In addition to this, Lim and Green (2008) showed that the perception of bitter taste and touch may be closely related. This may indicate that bitter taste also has a different

22 interaction with touch stimulation caused by tongue and mouth movements than other taste qualities.

The consideration of instructions regarding tongue and mouth movements, or

“tasting mode”, is particularly important when studying taste perception. Intraoral movements, which naturally occur when eating, combine with a number of sensations

(e.g. temperature) to form the overall impression of taste sensation (Gibson 1966). It is unclear how the failure to specify tasting mode may have affected findings in some previous studies on relative regional differences in taste responsiveness. Based on current knowledge of the effects of tongue and mouth movements on taste perception, enhancement or maintenance of perceived taste intensity is expected for all regions due to the fact that tongue and mouth movements prevent taste adaptation (i.e. the gradual decrease of taste sensation over time) (Theunissen et al., 2000). However, as previously mentioned, the degree of enhancement may be different on some regions of the tongue and for some taste qualities (see Spatial Summation above). The regional effects of tongue and mouth movements need to be investigated to understand how tasting mode may affect findings of regional differences.

1.6 STUDY OBJECTIVES

The current study had two major objectives 1) to investigate regional differences in taste responsiveness between carbohydrate stimuli (sucrose and MOS) and two prototypical taste stimuli, quinine and MPG, and 2) to investigate the impact of passive and active tasting modes on regional differences in taste responses. The comparison between carbohydrate stimuli might provide preliminary insight into the location or

23 function of a T1R2/T1R3 independent glucose oligomer sensing pathway in humans. The prototypical taste stimuli chosen (sucrose, quinine and MPG) were known to be perceived by G-protein coupled receptors (T1R2/T1R3; T2R; and T1R1/T1R3 or GluRs, respectively). MPG was selected to represent umami taste due to the lack of salty side taste. Taste stimuli were chosen to be approximately equi-intense on the tip of the tongue in passive tasting mode to investigate what impacts taste quality might have if stimuli intensity was similar.

The motivation for the second objective was two-fold. First, as was previously noted, many studies on regional differences in responsiveness do not specify tasting mode. However, studies on regional sensitivity have almost universally used some kind of passive tasting mode to prevent the spread of stimuli to other regions of the oral cavity.

Due to this omission in many studies using suprathreshold stimuli, it was deemed important to determine what effects an active tasting mode may have on relative regional differences, both to facilitate cross study comparisons and to determine whether it would be advisable to specify tasting mode in future studies.

Second, few studies have gathered data comparing the effects of tongue and mouth movement on stimulus applied to discrete regions of the oral cavity, and it is not currently known whether different regions of the tongue respond differently to intraoral tongue and mouth movements. However, it has been suggested that there may be a relationship between intraoral movement and umami taste, leading to a preferential enhancement of umami taste (Green and Nachtigal, 2012).

CHAPTER 2

REGIONAL DIFFERENCES IN TASTE RESPONSIVENESS: EFFECTS OF TASTE QUALITY AND TASTING MODE

Julie L. Colvin, Alexa J. Pullicin, Juyun Lim

Manuscript Submitted 05/25/2018

Chemical Senses

Regional Differences in Taste Responsiveness: Effects of Taste Quality and Tasting Mode

Julie L. Colvin, Alexa J. Pullicin, Juyun Lim

Department of Food Science and Technology, Oregon State University,

Corvallis, OR 97331, USA

Correspondence to be sent to: Juyun Lim, Ph.D.

Department of Food Science and Technology,

Oregon State University,

100 Wiegand Hall, Corvallis, OR 97331, USA

E-mail: [email protected]

Phone: +1-541-737-6507

Fax: +1-541-737-1877

Key words: Carbohydrate; Regional Differences; Taste; Tasting Mode; Umami

Running Head: Regional Differences in Taste Responsiveness

2.1 ABSTRACT

Previous studies have shown that there are differences in taste responses between various regions of the tongue. Most of those studies used a controlled ‘passive’ tasting mode due to the nature of investigation. However, food is rarely tasted in a passive manner. In addition, recent studies have suggested that humans can taste maltooligosaccharides (MOS) and that the gustatory detection of MOS is independent of the known sweet receptor. It is unknown whether regional differences in responsiveness to MOS exist and, if they exist, whether the pattern of the differences are parallel to that of simple sugars. This study was set up to investigate the effects of tasting modes

(‘passive’ vs. ‘active’) on regional differences in taste responsiveness to two carbohydrate stimuli, MOS and sucrose, as well as two other prototypical tastants, quinine and monopotassium glutamate (MPG). In the passive tasting condition, the tongue tip was found to be more responsive to both sucrose and MOS, but no regional differences were seen for quinine and MPG. In contrast, in the active tasting condition, the back of the tongue was found to be more responsive to quinine and MPG, but no differences were found of sucrose or MOS. These findings indicate that there are regional differences in taste responsiveness between the front and back of the tongue, and that regional responsiveness is dependent on taste quality and tasting mode.

2.2 INTRODUCTION

Since Hänig’s classic study (Hänig, 1901), it has been widely acknowledged that responses to tastes vary across the human tongue and other parts of the mouth (e.g., soft palate). However, there are only a limited number of studies supporting this notion, and further, there is no clear agreement about which regions are most sensitive to the four prototypical taste qualities (i.e., sweet, sour, salty, and bitter tastes; (Hänig, 1901;

Collings, 1974; Nilsson, 1979; Sato et al., 2002). Notably, all of these early investigations measured regional differences in taste responses in terms of thresholds, which cannot necessarily be generalized to taste responsiveness at suprathreshold levels (Keast and

Roper, 2007; Webb et al., 2015).

Studies of regional differences at suprathreshold levels have also been rare. An early example of one such study was conducted by Collings (1974), who reported that psychophysical functions for sour, salty, and bitter tastes differ between loci (i.e. front fungiform papillae, side fungiform papillae, foliate papillae, vallate papillae, and soft palate). More recently, Feeney and Hayes (2014) compared taste responsiveness on the anterior and posterior parts of the tongue for five taste qualities. They reported no regional differences for sweet, sour, and salty tastes, but significant differences for umami and bitter tastes; both were perceived as more intense on the posterior tongue than on the anterior tongue. Others have also measured responses to some or all of these taste qualities across various gustatory regions (Green and Schullery, 2003; Green and George,

2004; Green and Nachtigal, 2012; Doty et al., 2016), although the primary concern of these studies was not investigating regional differences in tastes responsiveness per se.

Nevertheless, the findings of these studies seem to generally agree with those of (Feeney and Hayes, 2014) with a few exceptions. For instance, some studies (Green and

Schullery, 2003; Green and George, 2004) found no significant difference in taste responsiveness to quinine between fungiform and circumvallate regions.

Interestingly, there are a wide range of methodological variations across these studies, which may contribute to differences in study findings. First, studies have employed various stimuli delivery techniques including filter paper disks (Collings, 1974;

Sato et al., 2002; Doty et al., 2016) and cotton tipped swabs (Green and Schullery, 2003;

Green and George, 2004; Green and Nachtigal, 2012; Feeney and Hayes, 2014). Second, the instructions given to subjects on mouth movement while evaluating stimuli (i.e.,

‘tasting mode’) range from passively keeping the mouth open (Nilsson, 1979a; Sato et al.,

2002; Doty et al., 2016) to actively pressing the tongue against the roof of the mouth and subsequently swallowing (Green and Nachtigal, 2012). Others do not describe the tasting mode used (Green and George, 2004; Feeney and Hayes, 2014; Green and Schullery,

2003). Importantly, an active tasting mode can spread a stimulus to other locations within the oral cavity and increase perceived intensity (Green and Nachtigal, 2012; Running and

Hayes, 2017). Third, the areas of the tongue where taste stimuli are delivered vary between studies. For example, Doty and colleagues (Doty et al., 2016) applied taste stimuli on each side of posterior tongue while others delivered stimuli onto the circumvallate papillae (e.g. Green and Schullery 2003; Feeney and Hayes 2014). Finally, the concentrations tested vary widely from threshold to suprathreshold levels and from low to high within suprathreshold levels. Relative differences between regions may be

29 concentration dependent as evidenced by the finding that slopes of psychophysical functions vary between loci (Collings, 1974).

The taste of fat (Running et al., 2015) and glucose oligomers (Lapis et al., 2014,

2016; Pullicin et al., 2017) have recently been suggested as potential novel taste categories. Accordingly, regional differences in fat taste have been investigated to better understand peripheral transduction mechanisms underlying the gustatory detection of free fatty acids (Mattes, 2009b); study findings showed no regional differences in detection threshold for any of the free fatty acids tested. However, the average intensity ratings for all free fatty acids tested were highest at the fungiform papillae followed by the circumvallate papillae and then the foliate papillae. In contrast, regional differences in taste responsiveness to glucose oligomers have not yet been investigated. An interesting study conducted by Vigorito et al. (1987) on rats reported that bilateral transection of the glossopharyngeal nerve, which innervates the posterior third of the tongue, reduced the consumption of Polycose (i.e., a glucose oligomer and polymer mixture) but not sucrose, while bilateral transection of the chorda tympani nerve, which innervates the anterior two thirds of the tongue, produced comparable reduction. Based on these findings, the authors suggested that while the detection of both classes of carbohydrates is mediated by multiple gustatory nerves, some of these nerves may have specialized functions.

The primary objective of the current study was to investigate regional differences in taste responsiveness to glucose oligomers, i.e., maltooligosaccharides (MOS), along with two other prototypical tastes. Sucrose was included to compare the regional differences of taste responsiveness between two classes of carbohydrate. As a comparison

30 to sweet taste, bitter and umami tastants also known to be transduced by G protein- coupled receptors (Hoon et al., 1999) were included. For bitter taste, one of the most common bitter compounds, quinine hydrochloride, was used. Monopotassium glutamate

(MPG) was included to represent umami taste instead of the more commonly-used monosodium glutamate (MSG); this decision was made because unlike MSG, MPG does not elicit significant salty taste (Maruyama et al., 2006; Chen et al., 2009; Green et al.,

2016). Given that the mode of tasting has been shown to modulate perceived intensities of tastes, in particular umami taste (Green and Nachtigal, 2012), we measured regional differences in taste responsiveness to the target stimuli using ‘passive’ and ‘active’ tasting modes in two separate experiments. In Experiment 1, a ‘passive’ tasting mode was used to minimize the spread of the stimulus to other regions of the tongue. After stimulus delivery, subjects were told not to allow their tongue to touch any part of their mouth by keeping their mouth open while rating. In Experiment 2, an ‘active’ tasting mode was used to mimic the motions that a subject might make during a normal eating condition.

2.3 MATERIALS AND METHODS

2.3.1 Subjects

Initially, a total of 31 subjects (17 F, 14 M) between the ages of 18 and 53 years of age (median: 26 years old) were recruited from Oregon State University and surrounding areas, and participated in the study. Data from 2 subjects were excluded due to localized taste insensitivity. One subject rated all stimuli below ‘barely detectable’ on one side of the tongue indicating potential damage to cranial nerves or some other condition (Tomita et al., 1986; Bartoshuk et al., 2012). Another subject had a thick white

31 film covering the back of the tongue, resulting in no perception on the region. Hence, data from 29 subjects (16 F, 13 M; median: 26 years old) were used for data analyses. A total of 26 subjects (14 F, 12 M) who participated in Experiment 1 returned for the

Experiment 2. Of those who participated, one subject could not follow the protocol (see below) due to difficulty simulating an active tasting motion. Therefore, data from 25 of the returning subjects (14 F, 11 M; median: 26 years old) were included in data analysis.

All subjects confirmed that they were healthy, non-smokers, not pregnant, and not taking prescription pain medication or insulin; had no history of taste or smell loss, or other oral disorders; had no oral lesions, canker sores, or oral piercings; and did not have a history of food allergies. Subjects were further asked to comply with the following restrictions prior to their testing session: 1) no consumption of food or beverage except water within

1 hour; 2) no use of any menthol-containing products within 1 hour, 3) no physically demanding activity within 1 hour, and 4) no consumption of spicy food on the day of testing. All subjects gave written consent and were compensated for their time. The experimental protocol was approved by the Oregon State University Institutional Review

Board and registered under the Clinical Trial registry (NCT02589353).

2.3.2 Stimuli

Five test stimuli were included in this study, prepared as aqueous solutions: 56 mM sucrose (Spectrum Chemical MFG Corp.), 0.10 mM quinine hydrochloride (Sigma

Aldrich), 0.32 M monopotassium glutamate (MPG; Ajinomoto), 224 mM maltooligosaccharide (MOS; average degree of polymerization of 14; prepared in (Balto et al., 2016), and a deionized water blank. The 4 taste stimuli were chosen to represent

32 sweet, bitter, savory/umami, and ‘starchy’ (Lapis et al., 2016) tastes, respectively. The water blank was included as a negative control. Stimuli concentrations were chosen to be approximately equi-intense when applied on the front of the tongue. In order to prevent

Figure 5 Diagram of the four target locations on a tongue.

oral enzymatic hydrolysis of MOS, this stimulus was prepared using an aqueous solution of 5 mM acarbose, a salivary α-amylase and α-glucosidase inhibitor (Clissold and

Edwards 1988; Balfour and McTavish 1993; Martin and Montgomery 1996). At this concentration, acarbose does not have a detectable taste, but is effective to prevent oral hydrolysis of MOS (Lapis et al., 2016). Test stimuli were applied to the targeted locations using cotton tipped swabs (see experimental procedure below). Saturated swabs contained approximately 0.20 mL of stimulus. All stimuli were stored at 4-6 °C and used within 1 week of preparation. Stimuli were served to subjects at room temperature (20-22

°C).

2.3.3 Experimental procedure

Experiment 1: Over 2 separate sessions, subjects were presented with a total of 20 trials comprised of the 5 test stimuli (4 taste stimuli and water control) applied in 4 targeted locations: the front and back of the left and right sides of the tongue (Figure 5). During each session, subjects received a total of 10 trials, which included all 5 stimuli applied on both the front and back of one side (left or right) of the tongue; the order of sides was counter-balanced across subjects. The presentation order of the 10 trials within each session was randomized across subjects. In addition, stimuli were presented on the front and back of the tongue in alternating order so that the same location would not be stimulated twice in a row. The location (front or back) of the first sample given was also alternated between sessions to ensure all subjects received both possible location presentation orders (i.e. back-front-back vs. front-back-front).

At the beginning of the first session, subjects were verbally instructed on the use of the general version of the labeled magnitude scale (gLMS; Green et al. 1993, 1996;

Bartoshuk et al. 2003). Subjects were asked to rate 15 remembered or imagined taste sensations (e.g., the sweetness of milk, and the burning sensation of eating a whole hot pepper) to familiarize themselves with making ratings over a broad context of sensations.

Following training on scale usage, a diagram of the 4 targeted tongue locations (see Fig.

5) was shown to subjects. Subjects were asked to visually locate the target locations on their own tongue using a mirror and to touch 2 of the targeted locations (i.e. front and back on one side) using a cotton swab saturated with water. This was done to minimize potential anxiety, discomfort, or gagging during swabbing, especially at the back of the

34 tongue. To familiarize subjects with the testing procedure, 2 practice trials were given using a water blank. During this practice trial, the experimenter swabbed 1 of the targeted regions of the tongue (e.g., front left) by rolling the saturated cotton swab across the target area 3 times (see Figure 6); this practice trial was repeated on the opposite region

(e.g., back left).

Figure 6 Picture of the front (left) and back (right) of the tongue being swabbed.

During each test trial, one of the 5 stimuli was applied on a targeted location following the swabbing procedure described above. Once a stimulus was applied to the tongue, subjects were asked to rate the peak taste intensity of the stimulus on the gLMS.

Importantly, subjects were instructed not to touch other areas of the mouth by keeping mouth slightly open while making ratings; this was done to minimize the spread of the stimulus to other regions of the tongue or mouth. Between each trial, subjects rinsed their

35 mouth at least 3 times with deionized water, then chewed on a 2-inch piece of plastic straw for 30 seconds to encourage saliva production. Subjects were instructed to chew the straw at the same rate that they naturally chew food. This procedure was found to effectively reduce carry over tastes between stimuli and also to counter dryness in the mouth after repeated rinsing. Subjects were given an additional 30 second break between trials and a 3 minute break after half of the trials were presented.

Experiment 2: Experimental design was consistent with that described in the previous experiment except subjects were told to taste samples using their natural ‘active’ tasting motion. This motion was described as being similar to how they may taste a new food or beverage for the first time; for example, it may be a smacking motion. The subjects were asked to practice active tasting by mimicking their natural tasting motion 3 times.

Importantly, subjects were instructed not to swallow while tasting or rating samples, as swallowing has been shown to increase the perceived intensity of bitter taste (Running and Hayes, 2017). In addition, subjects were asked to open their mouth immediately after completing 3 tasting motions and to keep their mouth open while rating the peak taste intensity of the stimulus on the gLMS; this was done to prevent continued contact between the tongue and the rest of the mouth while rating. To familiarize subjects with the test procedure, subjects performed 2 practice trials using the water control as described in Experiment 1, but with the inclusion of the active tasting motion.

2.3.4 Data analysis

All data collected were log transformed before any statistical analysis, as gLMS responses tend to be log-normally distributed (Green et al., 1993, 1996). Repeated-

36 measures analysis of variance (ANOVA) was first performed on taste ratings using stimulus, region (front vs. back) and side (left vs. right) as factors. Sex was also included as a predictor in the model. While stimuli and region were found to be significant, side and sex alone were not found to be significant (p > 0.05). Therefore, data obtained from each side were treated as replicates and ratings were averaged across replicates. To further analyze the effects of stimuli and tongue region on the perceived intensities, a repeated measures ANOVA was conducted again using averaged data. Post-hoc paired t- tests were conducted to analyze regional differences within each stimulus. In addition, regional differences in taste responsiveness were compared between active and passive tasting modes for subjects that completed both experiments. To determine how tasting mode impacts the regional differences in taste responsiveness of the stimuli, a repeated measures ANOVA was conducted on averaged data (left and right replicates) using tasting mode, region, and stimulus as factors. Statisica 8 (Stat Soft, Inc.) was used for all statistical analysis.

2.4 RESULTS

2.4.1 Experiment 1: passive tasting

Figure 7 displays the log mean ratings of all 5 stimuli on 2 different tongue regions: front (gray bars) and back (black bars). Mean perceived intensities for the 4 taste stimuli were rated around ‘weak’, while the water control was rated around ‘barely detectable’. Repeated measures ANOVA revealed that stimuli [F(4,112) = 25.90, p <

0.0001], region [F(1,28) = 9.77, p < 0.005] and the interaction between region and stimuli [F(4,112) = 5.99, p < 0.0005] had significant effects. To further investigate

37 regional differences in taste responsiveness, paired t-tests were performed for each stimulus. As expected, there was no regional difference in perceived intensity for the water control (t-value = -0.11, p > 0.05). Surprisingly, regional differences in perceived intensity for quinine and MPG were not found to be significant (t-values: -1.02 and 1.33, with p > 0.05, respectively). However, both sucrose and MOS were perceived as significantly stronger on the front of the tongue (t-values: 3.77 and 4.69, p < 0.001 and <

0.0001, respectively).

Figure 7. The mean log perceived intensities of stimuli sampled on the front and back using a passive tasting mode are displayed. Bars represent standard error. Left Y-axis represents log perceived taste intensity. The right Y-axis represents the semantic labels of the gLMS: BD = barely detectable, W = weak, M = moderate, and S = strong. * indicate significant differences between mean perceived intensity ratings performed by paired t-tests.

2.4.2 Experiment 2: active tasting

Figure 8 displays the log mean ratings of all 5 stimuli on 2 different tongue regions: front (gray bars) and back (black bars). Repeated-measures ANOVA revealed that stimuli [F(4,96) = 35.09, p < 0.0001] and region [F(1,24) = 6.26, p < 0.05] had significant effects. The interaction effect between region and stimuli [F(4,96) = 3.49, p <

0.05] was also found to be significant. Post hoc paired t-tests were performed to further investigate regional differences for each stimulus. The water control was found to be perceived as stronger on the back of the tongue, but this difference fell short of significance (t-value = -1.96, p = 0.06). Unlike Experiment 1, no significant regional differences were found for sucrose and MOS (t-values: -0.27 and 0.61, with p > 0.05).

However, quinine and MPG were found to be perceived as significantly stronger on the back of the tongue (t-values: -2.92, and -3.88, p < 0.01 and < 0.001, respectively).

2.4.3 Impact of tasting mode on regional differences in taste responsiveness across stimuli

In order to directly compare the impact of tasting mode on the regional differences observed across the stimuli tested, repeated-measures ANOVA was conducted with tasting mode, region, and stimulus as factors. The analysis revealed that tasting mode and region were not significant factors alone (p > 0.05), but stimulus was significant [F(4,96) = 37.17, p < 0.0001], though the latter can be attributed to the inclusion of the blank stimulus. The interaction effects between mode and region [F(1,24)

= 21.87, p < 0.0001], mode and stimuli [F(4,96) = 2.78, p < 0.05], and region and stimuli

[F(4,96) = 7.52, p < 0.0001] were significant. These results suggest that the mode of

39 tasting and region did not vary in a systematic matter, but that the impact of tasting mode differed across the regions.

Figure 8. The mean log perceived intensities of stimuli sampled on the front and back using an active tasting mode are displayed. Bars represent standard error. Left Y-axis represents log perceived taste intensity. The right Y-axis represents the semantic labels of the gLMS: BD = barely detectable, W = weak, M = moderate, and S = strong. * indicate significant differences between mean perceived intensity ratings performed by paired t-tests.

2.5 DISCUSSION

The results from the current study generally support the notion that responsiveness to taste varies across gustatory regions. More notably, our results indicate that regional differences depend on taste quality and tasting mode. While the “tongue map” is the most widely known example of regional differences in taste perception, it has become clear that the premise was likely based on a misinterpretation of Hänig’s (1901) classic study (Bartoshuk and Beauchamp, 1994). The major critique of the “tongue map”

40 is that it implies localized regions of the tongue are solely responsible for eliciting each taste quality (Breslin, 2001). However, Hänig’s original findings indicate that taste sensitivity varies across the tongue in a quality dependent manner; for example, the tip of the tongue was found to be slightly more sensitive to sweet taste than the rest of the tongue.

2.5.1 Regional Differences in Taste Responsiveness: Taste Quality

In the current study, both carbohydrate stimuli, sucrose and MOS, were perceived as more intense on the front than the back of the tongue when a passive tasting mode was employed (see Fig. 7). This result is in contrast with the recent findings (Green and

Schullery, 2003; Green and George, 2004; Green and Nachtigal, 2012; Feeney and

Hayes, 2014), which found no regional difference in the perceived intensity of sweetness.

One noticeable difference between those studies and the current study is stimulus concentration; while those studies used 0.18 to 2.0 M (6.2 to 68.5 %) sucrose, the current study used 56 mM (1.9 %) sucrose. Recall that Collings (1974) found psychophysical functions vary between loci. Importantly, studies using a similar concentration range have reported that the front of the tongue is more sensitive to sucrose than the back of the tongue in terms of taste detection threshold (Hänig, 1901) or quality recognition threshold (i.e. subjects correctly identify sucrose as “sweet”; (Collings, 1974; Nilsson,

1979). Combined together, it appears that at relatively lower concentrations, the tip of the tongue is more responsive than the back of the tongue to carbohydrate stimuli, and this difference may subside as concentration increases.

For umami taste, previous studies have reported a marked degree of regional differences in responsiveness to monosodium glutamate (MSG) or a mixture of MSG and inosine monophosphate (IMP), with responses being considerably stronger on the back of the tongue (Yamaguchi and Ninomiya, 2000; Green and Nachtigal, 2012; Feeney and

Hayes, 2014). In contrast, the current study showed the front and back of the tongue were equally responsive to MPG, with a slight trend of the front being more responsive, when a passive tasting mode was employed (see Fig. 7). Notably, the concentrations tested were fairly comparable between the current (0.32 M) and previous studies (0.08 – 0.25

M). One possible explanation could be the differences in the stimuli tested. While MSG is commonly used as a prototypical umami substance, it has a significant “salt- dependent” gustatory component (Maruyama et al., 2006; Chen et al., 2009; Green et al.,

2016). Accordingly, the qualitative similarity between MSG and NaCl at the above mentioned concentrations has been noted previously (Yoshida and Saito, 1969), along with a possible confusion between saltiness and umami (Green et al., 2016). More interestingly, Ninomiya and Funakoshi (1989) demonstrated using a mouse model that the afferent input from the glossopharyngeal nerve, which innervates the posterior tongue, plays a more significant role for behavioral discrimination between MSG and

NaCl than that from the chorda tympani nerve, which innervates the anterior tongue; the authors speculated that the glossopharyngeal nerve conveys relatively more information of the anion (glutamic acid) than the cation (sodium) component of MSG, while the reverse is true for the chorda tympani nerve. Potential differences in the detection mechanism for MSG and MPG are further supported by more recent studies. It has been

42 shown that lactisole (i.e., a sweet taste blocker), which binds to the transmembrane region of T1R3 (Jiang et al., 2005), can also significantly suppress the umami taste of MSG

(Galindo-Cuspinera and Breslin, 2006), but not MPG (Green et al., 2016). These study findings together suggest that transduction mechanisms may differ between MSG and

MPG, which can explain the disagreement in the reported regional differences for the two umami substances. The concept that stimuli representative of the same taste quality (e.g., aspartame and sucrose) exhibit different spatial profiles has been acknowledged previously (Breslin, 2001).

For bitterness, the current study found the front and back of the tongue to be similarly responsive to quinine with a non-significant trend for elevated responsiveness on the back of the tongue, when a passive tasting mode was employed (see Fig. 7).

Previous studies have also found similar results and trends for the responsiveness to quinine (Green and Schullery, 2003; Green and George, 2004), although one study reported the back of the tongue being significantly more responsive (Feeney and Hayes,

2014). It should be noted that the latter study used a higher concentration (2 mM) of quinine than any other studies (0.18-1 mM) including the current (0.1 mM), which suggests regional differences in responsiveness to bitter taste may also be affected by stimulus concentration. This possibility gains support from the study of Collings (1974), which showed bitter taste intensified more quickly on the back of the tongue as a function of concentration. Therefore, while the back of the tongue is generally more responsive to bitter taste, variation in concentration may have affected the degree of differences shown between the regions.

2.5.2 Regional Differences in Taste Responsiveness: Tasting Mode

The pattern of regional differences in taste responsiveness differed depending on the tasting mode. When a passive tasting mode was employed, the front of the tongue was more responsive to sucrose and MOS than the back, while the two tongue regions were equally responsive to quinine and MPG (see Fig. 7). In contrast, when an active tasting mode was employed, both regions were equally responsive to sucrose and MOS, while the back of the tongue was more responsive to quinine and MPG (see Fig. 8). The comparison of these findings demonstrate that regional differences in taste responsiveness depend on not only taste quality but also tasting mode. A careful inspection suggests that the adaption of an active tasting mode decreased the perceived intensities of all stimuli on the front of the tongue, but increased the perceived intensities of all stimuli, except quinine, on the back of the tongue. This finding is illustrated in

Figure 9. When the passive tasting mode was employed, subjects were instructed to keep their tongue from touching other parts of the mouth by keeping the mouth slightly open.

During the active tasting trial, subjects were asked to mimic their own natural tasting motion, which was described as “the motion you would naturally make when tasting a new food or beverage; for example, a smacking motion”. Subjects were also asked not to swallow after making the active tasting motion. Given the natural variation in the extent and pattern of tongue and mouth movements made during this active tasting motion, the impact on taste responsiveness could have differed slightly across subjects. However, considering the steps of typical oral processing of food (de Wijk et al., 2003), subjects likely pressed the tongue against the palate and employed a tongue movement of some

Figure 9. The mean differences in log perceived intensities between passive and active tasting modes are displayed. Differences between modes were calculated by subtracting the log perceived intensities of each stimulus obtained under the passive tasting mode from those under the active tasting mode. The means of those differences were calculated for all stimuli on each region. Right Y-axis represents differences (active - passive) in log perceived taste intensities between the two tasting modes. kind. Accordingly, when a motion like this is made, stimuli applied on the tongue tip are likely to be spread to relatively insensitive regions such as the hard palate (Nilsson,

1979a; Green and Nachtigal, 2012) and medial tongue (Doty et al., 2016), which would result in a decrease in responsiveness. In contrast, when stimuli are applied to the back of the tongue, the tongue and mouth movement can spread stimuli to the soft palate, a sensitive gustatory region (Collings, 1974; Nilsson, 1979a; Sato et al., 2002). We speculate that the spread of stimuli from the tongue to the soft palate increased the area of gustatory stimulation, and consequently, increased perceived intensity due to spatial summation (Smith, 1971; Delwiche et al., 2001a). Notably, the perceived intensity of

MPG was increased by active tasting on the back of the tongue more than any other taste stimulus. A similar trend was noted in previous work on the effects of tongue and mouth movement on the responsiveness to MSG (Green and Nachtigal, 2012).

2.5.3 Regional Taste Responsiveness to Carbohydrate Stimuli

Recent studies in our lab have shown that humans can taste MOS and that their gustatory detection is independent of the hT1R2/hT1R3 sweet receptor (Lapis et al.,

2016; Pullicin et al., 2017). The current study investigated the regional differences in taste responsiveness to MOS in human subjects for the first time. Our results suggest that the tongue tip is more responsive to MOS than the back of the tongue under the passive tasting mode, but that both areas are equally responsive when the active tasting mode is employed (see Fig 7 and 8). These findings are not necessarily in good agreement with one available study; Vigorito et al. (1987) reported that bilateral transection of the glossopharyngeal nerve in rats reduced the consumption of Polycose but not sucrose, while bilateral transection of the chorda tympani nerve produced comparable reduction.

Note Polycose is a mixture of maltooligo- and maltopoly-saccharides (MOS and MPS).

Based on these findings, the authors hypothesized that stimulating the posterior tongue with MOS/MPS would produce a stronger response than stimulating the same region with sucrose. However, we found no differences between the responses to MOS and sucrose on either the front or the back of the tongue. It is difficult to explain these conflicting results at this time, although we speculate that differences between species along with experimental procedures (e.g., chemical specificity of stimuli, the control of salivary amylase) may have caused the discrepancy. Further investigation is warranted to

46 determine the sensory mechanisms underlying the gustatory detection of glucose oligomers in humans.

2.6 SUMMARY

The current findings suggest that regional differences in taste responsiveness depend on stimuli quality and concentration, and further that the adaption of tasting mode modulates responsiveness in a region specific manner. It has been suggested that the regional differences in taste responses could have a functional purpose; while the primary function of the anterior tongue may be stimulus discrimination, the role of the posterior tongue may be to promote acceptance vs. rejection of the stimulus (Breslin, 2001). While the current study did not intend to investigate functional differences between gustatory regions, we speculate that some of the regional differences we observed may fit in this notion. For example, the heightened responsiveness to MOS and sucrose on the tongue tip, in particular when the more controlled passive tasting mode was used, may encourage the consumption of carbohydrates even at low concentrations (e.g., porridge). To the contrary, relatively heightened responsiveness to quinine and MPG on the back of the tongue may serve to promote rejection and acceptance of its consumption, respectively.

The fact that responsiveness to MPG was increased the most by active tasting is also intriguing given that savory foods, like cooked meat (Breslin, 2013), require chewing.

Taken together, the regional differences demonstrated in the current study may have developed to serve the gustatory functions of selecting and subsequently accepting/rejecting potential foods that are complex in nature.

2.7 REFERENCES

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Bartoshuk LM, Catalanotto F, Hoffman H, Logan H, and Snyder DJ. 2012. Taste damage (otitis media, tonsillectomy and head and neck cancer), oral sensations and BMI. Physiol Behav. 107:516–526.

Bartoshuk LM, Duffy VB, Fast K, Green BG, Prutkin J, and Snyder DJ. 2003. Labeled scales (e.g., category, Likert, VAS) and invalid across-group comparisons: what we have learned from genetic variation in taste. Food Qual Prefer. 14:125–138.

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Chen Q-Y, Alarcon S, Tharp A, Ahmed OM, Estrella NL, Greene TA, Rucker J, and Breslin PAS. 2009. Perceptual variation in umami taste and polymorphisms in TAS1R taste receptor genes1234. Am J Clin Nutr. 90:770S-779S.

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Delwiche JF, Buletic Z, and Breslin PAS. 2001. Relationship of papillae number to bitter intensity of quinine and PROP within and between individuals. Physiol Behav. 74:329– 337.

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Green BG, Dalton P, Cowart B, Shaffer G, Rankin K, and Higgins J. 1996. Evaluating the ‘Labeled Magnitude Scale’ for Measuring Sensations of Taste and Smell. Chem Senses. 21:323–334.

Green BG, and George P. 2004. ‘Thermal Taste’ Predicts Higher Responsiveness to Chemical Taste and Flavor. Chem Senses. 29:617–628.

Green BG, and Nachtigal D. 2012. Somatosensory factors in taste perception: Effects of active tasting and solution temperature. Physiol Behav. 107:488–495.

Green BG, and Schullery MT. 2003. Stimulation of Bitterness by Capsaicin and Menthol: Differences Between Lingual Areas Innervated by the Glossopharyngeal and Chorda Tympani Nerves. Chem Senses. 28:45–55.

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CHAPTER 3

GENERAL CONCLUSIONS

The objectives of this study were 1) to investigate the effects of taste quality on regional differences in taste responsiveness, and 2) to investigate the effects of passive and active tasting modes on regional differences in taste responses.

For the first objective, we found that taste quality impacted regional differences.

However, both carbohydrate stimuli (sucrose and MOS) demonstrated the similar patterns of regional differences in both tasting modes. While this contradicts our hypothesis and previous findings, studies on the regional differences for “olegustus” (i.e., the taste of fatty acids Running and Hayes, 2017) have also reported unexpected patterns of regional differences given the current knowledge about taste perception mechanisms

(Mattes, 2009b). Regional differences for quinine were generally in agreement with the findings of previous studies, especially considering the proposed concentration effects.

Findings for MPG were unexpected. Considering the differences in perceptual attributes and transduction mechanisms discussed in Chapters 1 and 2, this may indicate that the salty component of MSG has an enhancement effect on the back of the tongue or that discrimination of umami taste is different between tongue regions. These findings highlight the need for more research into transduction methods of non-prototypical taste stimuli and the use of a greater diversity of chemical stimuli, even when studying prototypical taste qualities, to better understand transduction mechanisms and their relationship with regional perception.

For the second objective, we found that tasting mode modulated regional differences in taste responsiveness for all stimuli. This highlights the importance of controlling for tasting mode in studies on relative regional differences. Further, we found that changes in regional differences arose from region specific effects of tasting mode.

On the front of the tongue, perceived intensity decreased for all stimuli. This was unexpected considering previous findings indicating an active tasting mode would increase perceived intensities on all regions and that an active tasting mode would prevent taste adaptation (i.e. decreases in perceived intensity over time). These differences may have been due to the methods of stimulation or other variation in methodology. However, on the back of the tongue, there was a general increase in intensity for all stimuli except quinine. Finally, our results show that umami taste elicited by MPG, increased more than other stimuli during tongue and mouth movements. This generally agrees with previous findings on the enhancement interaction between umami taste and tongue and mouth movements (Green and Nachtigal, 2012). However, it suggests that central mechanisms for the integration of touch and taste information may be more robust for the back of the tongue. This makes sense from a functional stand point, as food is typically exposed to intraoral processing before it reaches the back of the tongue.

In summary, differences in tongue anatomy and other perceptual mechanisms may contribute to the effects of tasting quality and tasting mode on differences in responsiveness between tongue regions. Further, these differences may have some adaptive purpose beyond protective redundancy.

This research improves the overall understanding of regional differences in taste responsiveness between tongue regions. The importance of controlling for mouth and tongue movements while measuring regional differences in taste responsiveness is emphasized by the finding that relative regional differences changed depending on the tasting mode used. This is particularly important because some reports do not detail the instructions given to subjects on tongue and mouth movements. These results also suggest that the taste stimuli used to elicit each taste quality (i.e. MPG vs MSG) and the intensity of taste stimuli are important considerations when designing a study on regional differences. From a clinical perspective, the differences between the effects of tongue and mouth movements on the front and back of the tongue may help to better understand the consequences of localized taste loss. And finally, understanding the perception of taste stimuli on different regions of the tongue may help tailor the “spatial profile” of foods. In other words, this may help to formulate foods so that some taste qualities are experienced more strongly on the front or back of the tongue by using different taste stimuli.

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